Hepatitis C virus (HCV) remains an urgent global threat, with increasing rates of new infections in many jurisdictions due to a resurgence of injection drug use. People who inject drugs (PWID) and MSM are also at risk for HIV infection and when co-infected with these two viruses they suffer from accelerated liver disease progression and lower treatment response rates when interferon-based therapies were used. New interferon- sparing therapies with direct-acting antivirals (DAAs) lead to cure rates of over 95%, even in HCV/HIV coinfected persons. However, there remains an urgent need to protect high-risk populations from chronic HCV infection and re-infection as treatment alone might be insufficient in these populations with often limited access to health care. Prophylactic interventions will require a better understanding of the immune response necessary for protection from chronic infection and how cured subjects can be prevented from re-infection, with special considerations of additional challenges in the context of HIV co-infection and substance use. In this proposal we focus on the impact of HIV co-infection and of drug abuse on the generation and recovery of HCV- specific T cells during primary infection and after therapy. We will utilize PBMC from well-defined cohorts of persons with acute and chronic HCV/HIV co-infection, including longitudinal samples from patients undergoing DAA therapy, together with direct ex-vivo analysis of HCV-specific CD4 and CD8 T cells by flow cytometry and RNAseq of sorted cells on the single cell and population level. Specifically we will define the impact of HIV co-infection on the quality of the critical CD4 T cell response generated during primary HCV infection in HIV positive hosts, will identify the mechanism enabling sustained and functional HCV-specific CD4 responses in patients receiving early DAA treatment, and will determine whether HIV co-infection and substance abuse impair the recovery of HCV-specific T cells after DAA therapy for chronic HCV infection. The results will deliver important insights into the pathogenesis of HCV infection and will inform immunological approaches for the prevention of chronic HCV infection and re-infection in high risk patients with HIV co-infection and/or substance use.